Japan has already approved importing and selling 50 or more varieties of genetically modified crops (hereinafter, referred to as “GMOs”) such as corn, soybeans, and potato that have passed safety assessment. In connection with this, GMO-containing foods must be labeled in accordance with “the Labeling Standards for Genetically Modified Foods established by the Ministry of Agriculture, Forestry and Fisheries based on Article 7, paragraph 1 of the Quality Labeling Standard for Processed Foods and Article 7, paragraph 1 of the Quality Labeling Standard for Fresh Food” (Notification No. 517 of the Ministry of Agriculture, Forestry and Fisheries, Mar. 31, 2000) and “the Enforcement of Ministerial Ordinance amending in part the Ministerial Ordinance on Food Sanitation Law Enforcement Regulations and Compositional Standards, etc. for Milk and Milk Products” (Notice No. 79 of the Food Sanitation Department, Ministry of Health, Labor and Welfare, Mar. 15, 2001).
In other countries, however, GMOs may be cultivated in some cases together with non-GMOs once the safety evaluation thereof has been completed, or contamination may occur during the process of distribution after harvest. Moreover, many food processors and the like often contract the manufacture of processed foods to manufacturing companies, and even if they stipulate that non-GMOs should, be used, if GMOs are used in the plants of the manufacturing companies, small quantities of such GMOs may contaminate processed foods. Consequently, in order to fulfill their labeling obligations, food processors and the like must assess and analyze the final processed food products to confirm that they are not contaminated by GMOs.
Known methods of detecting GMOs in test samples of processed foods, raw materials thereof, etc. include a method of detecting a recombinant DNA by polymerase chain reaction (hereinafter, referred to as “PCR”) and a method of detecting a recombinant protein by an enzyme-linked immunosorbent assay (hereinafter, referred to as “ELISA”). In the case of processed foods, many proteins are denatured due to heating or pressurizing and cannot be, thereby, correctly detected by ELISA in many cases. Consequently, detection by PCR is commonly performed.
Known methods for assessment and analysis of GMOs include the method described in “the JAS Analytical Handbook, Manual of Assessment and Analysis for Genetically Modified Foods, Revised Second Edition” and the method described in “Concerning Testing Methods for Foods Modified by Recombinant DNA Technology (Partially Revised)” (Notice No. 0618002 of the Food Sanitation Department, Ministry of Health, Labor and Welfare, Jun. 18, 2003). These describe that in the testing and analysis of GMOs, in order to confirm whether DNA extracted from a test sample can be amplified by PCR, it is necessary to confirm whether a PCR product having an expected length is obtained by PCR using a primer pair recognizing a species-specific DMA of each agricultural product. In quantification of a GMO contained in a test sample, the contamination rate of a recombinant is relatively determined based on the abundance ratio of a recombinant DNA to a species-specific DNA that is always present in the crop.
For example, in the case of corn, primer pairs that specifically recognize respectively the five lines of approved GMO strains have been developed, and also a primer pair that recognizes the SSIIB gene region has been developed as a corn species-specific DNA.
Because this primer pair that recognizes the species-specific DNA provides a standard for the amount of the species-specific DNA in recombinant DNA detection and quantification, the region of the species-specific DNA to be amplified should be present in a single copy on the genome. In “Concerning Testing Methods for Foods Modified by Recombinant DNA Technology (Partially Revised)” (Notice No. 1113001 of the Food Sanitation Department, Ministry of Health, Labor and Welfare, Nov. 13, 2003), quantitative PCR is performed using a standard substance that is an amplification product amplified by a specific primer pair targeting a corn or soybean species-specific DNA and a recombinant DNA and linked to a plasmid. The ratio of the number of copies of the recombinant DNA to the number of copies of the species-specific DNA can be accurately determined by performing the quantitative PCR using this standard substance and also performing the quantitative PCR of a test sample for a predetermined time.
When there are multiple lines of GMO strains, as in corn, use of a standard substance where a DNA specific to any line and a species-specific DNA are linked to a single circular DNA usefully allows use of a common standard substance in measurement of the contamination rate of any line.
It is generally difficult to obtain a gene specific to any line, but once a replicable DNA containing it has been prepared, a line-specific DNA can be also stably obtained by replicating this DNA itself.